1. Field of the Invention
The present invention relates generally to the field of reporter genes and particularly provides improved green fluorescent protein (GFP) genes, constructs and methods of use. The gfp genes disclosed herein are humanized gfp genes adapted for expression in mammalian and human cells by using preferred DNA codons.
2. Description of the Related Art
Reporter molecules are frequently used in biological systems to monitor gene expression. Commonly used reporter genes include .beta.-galactosidase, firefly luciferase, alkaline phosphatase, chloramphenicol acetyltransferase (CAT) and .beta.-glucuronidase (GUS). However, the available reporter genes have certain drawbacks that limit their use. A frequently encountered limitation is that the introduction of a substrate is required. Other drawbacks include, for example, the size of certain proteins which means that expression of reporter-fusion proteins can be difficult.
Another useful strategy is to label a protein with a fluorescent tag to enable subsequent detection and localization in intact cells. Fluorescent labeling is used in conjunction with immunofluorescence and fluorescence analog cytochemistry, in which the biochemistry and trafficking of proteins are monitored after microinjection into living cells.
Fluorescence labeling has generally been achieved by purifying proteins and covalently conjugating them to reactive derivatives of organic fluorophores. In these methods, the stoichiometry and locations of dye attachment are often difficult to control and careful repurification of the proteins is usually necessary. A further problem is introducing the labeled proteins into a cell, which often involve microinjection techniques or methods of reversible permeabilization to introduce the proteins through the plasma membrane.
A molecular biological alternative to fluorescent-tagged proteins has been made possible by recent advances and the cloning of green fluorescent protein (GFP). The green fluorescent protein (GFP) encoded by the gfp 10 gene from the jellyfish Aequorea Victoria is a protein of 238 amino acids which absorbs blue light (major peak at 395 nm) and emits green light (major peak at 509 nm) (Morin and Hastings, 1971; Ward et al., 1980; Prasher et al., 1992). The GFP hexapeptide chromophore starts at amino acid 64 and is derived from the primary amino acid sequence through the cyclization of serine-dehydrotyrosine-glycine within this hexapeptide (Shimomura, 1979; Cody et al., 1993).
The light-stimulated GFP fluorescence is species-independent and does not require any cofactors, substrates, or additional gene products from A. Victoria (Chalfie et al., 1994). This allows GFP detection in living cells other than A. Victoria so long as meaningful gene expression can be achieved. The small size of gfp 10 and the "real-time" detection of the product thus makes GFP a promising candidate for use as a reporter gene.
Certain GFP variants have recently been reported that have improved spectral properties. For example, Heim et. al. (1994) described a mutant that fluoresces blue and contains a histidine in place of Tyr66. Heim et. al. (1995) later described a Ser65.fwdarw.Thr GFP mutant that has a spectrum much closer to that of Renilla reniformis, which has an extinction coefficient per monomer more than 10 times that of the longer-wavelength peak of Aequorea GFP.
However, despite certain developments, such as the variants described above, the present usefulness of GFP is still limited by variable and, at best, low expression levels in mammalian cells. Therefore, it is evident that new developments in GFP technology are needed before the full potential of this protein can be realized, particularly in applications that require expression in mammalian cells, including gene therapy strategies.